MDR Function: 4 Prior Treatment: yes p53 Status: unknown Age: 11 BioSourceType: other[unknown] CellLine: SR DiseaseState: Lymphoma Individual: InitialTimePoint: birth OrganismPart: blood Sex: male TargetedCellType: Lymphoma TimeUnit: years
Growth protocol
NCI60Suspended; RNA harvesting protocol for suspended cells: Media used: RPMI 1640 500 ml FBS 25 ml 200 mM Glutamine 5 ml Cells: start from growing cells from Frederick (not frozen). Started before at passage #8-12. Do not use past passage 20. Grow 10 flasks. 1 flask = ~15 x 106 cells yields ~ 100 ugr RNA. Growth schedule prior to harvest Count cells as necessary to follow growth. Grow cells to ~0.52 x 106 cells /ml Pass 1x106 cells into each T162 w 30 ml media. Pass cells to as many flasks as there are cells for. When passing cells, combine into a single pool. Repeat growth cycle until 10 flasks are available. Refeed Refeed cells the day prior to harvest (without passsing). To do so: Spin down cells 1.7k rpm, 10 min., in a 50 ml tube. Draw off media. Resuspend in 30 ml media. Return to flask. Harvest Harvest at 0.52 x 106cells per ml when 10 flasks are available. Spin 4 flasks of cells at 1.7k rpm (~ 500 x g), 10 min (speed setting at 3.5), in a 250 ml centrifuge tube. Draw off media, leaving ~0.5 ml behind. Flick centirifuge tube to break up cells. Lyse cells in 15 ml lysis buffer (w 10 ul fresh BME per ml) per 4 T162âs. Vortex 10 sec. Draw thru a 20 guage needle 12xâs. Freeze at ö800C. Purify using Quigen Midi Kit.Use a maximum of 100 x 106 cells per column. More will not bind to the column. Company Info: Fetal bovine serum Do not heat inactivate. Bio Whittaker Cat# 14-501F Lot #9S083F 301-898-7025 Quiagen Midi Kit Cat # 75144 DTT Fluka or Gallard 1x PBS pH 7.3-7.5 Bio Whittaker cat #17-516F $5.30 per 500 ml bottle (1-11) 250 ml conical centrifuge tubes Corning cat # 25350-250 RPMI-1640 wo L-glutamine cat # 12-167F $13.25 per 500 ml bottle (for 1-11 bottles) Cambrex (old Bio Whittaker) Walkersville, Md. 21793 301-898-7025 1-800-638-8174 Trypsin (0.05%)-EDTA (0.1%) In phosphate buffered saline without calcium and magnesium. cat # 118-087-061 Quality Biological, Inc. 301-840-9331 L-glutamine 200 mM, 100x Gibco-BRL cat # 25030-081 162 cm2 flask cat # 3150 Costar Tissue Culture Cell Scrapper, 25cm Sarstedt Cat. # 83.1830 Cells are recieved from Nick Scudiero, E-mail:SCUDIERO@dtpax2.ncifcrf.gov Also trypsin, FBS and glutamine have been coming from there as well.; Protocol Type = grow; Parameter start Time = 0; Parameter min temperature = 37; Parameter media = RPMI 1640,5% FBS, 1% L-Glutamine;
Extracted molecule
total RNA
Extraction protocol
NCI60 Extraction Protocol; Harvest Target confluency 80% # of flasks = 10 Draw off media from 1st flask. Lyse cells in 15 ml lysis buffer (w 10 ul fresh BME per ml). Scrape cells. Draw off media from 2nd flask. Pipet lysis buffer from flask 1 into flask 2. Repeat lysis with up to 4 T162’s. Pipet into 50 ml tube. Repeat w next set of 4 flasks. When done, vortex 10 sec.. Draw lysate thru a 20 guage needle 12x’s. Freeze at –800C. Purify using Quiagen Midi Kit. Use a maximun of 100 x 106 cells per column. More will not bind to the column. ; Protocol Type = nucleic_acid_extraction; Parameter Extracted product = total RNA; Parameter Amplification = none;
